KR20070004821A - Method and system for tomographic imaging using fluorescent proteins - Google Patents

Abstract

A system for optical tomography includes an apparent light source adapted to project excitation light toward a specimen having fluorescent proteins therein, wherein the excitation light enters the specimen becoming intrinsic light within the specimen, wherein the intrinsic light is adapted to excite fluorescent light from the fluorescent proteins, and wherein the intrinsic light and the fluorescent light are diffuse. A method of optical tomography includes generating the excitation light with the apparent light source, wherein the intrinsic light and the fluorescent light are diffuse. ® KIPO & WIPO 2007

Description

Method and system for tomography imaging using fluorescent protein {METHOD AND SYSTEM FOR TOMOGRAPHIC IMAGING USING FLUORESCENT PROTEINS}

FIELD OF THE INVENTION The present invention generally relates to optical tomography, and more particularly, to methods and systems for extracting quantitative, three-dimensional molecular and biological information from living samples using fluorescent proteins.

Fluorescent proteins (FPs) are important reporter molecules in various biomedical applications. In some current applications, the treated fluorescent proteins are detected by surface fluorescence, confocal microscopy or reflection (whole animal) images and the like.

Surface fluorescence and confocal microscopy depend on coherent (non-dispersive) light that is projected onto and reflected from the sample. Because the microscope requires a significant amount of coherent light, this technique can only image up to the shallow depth of the sample (eg, about 1 nm or less). In the case of deeper imaging, light is known to diffuse and the microscope becomes inefficient for deeper imaging.

Reflective fluorescence imaging has been known to be useful for detecting and tracking cancers, particularly cancers occurring on surfaces or surgically exposed organs. However, the reflected fluorescence image has a fundamental limitation, and since the obtained image is a superposition of fluorescence signals generated at various depths, it tends to cause blurry images. Also, the reflected fluorescence image is not tomography, and only absolute quantification of fluorescence activity is possible without giving information about depth. This is partly due to the attenuation and amplification of nonlinear light in biological tissues, which limits the application of reflective fluorescence imaging to semi-quantitative imaging only at depths of a few millimeters.

Imaging of optical signals in deeper tissues often requires improved optical excitation and application of detection devices and techniques, and the use of tomography principles to combine data obtained from various projections.

Advances in imaging with diffractive light sources have led to a number of studies examining tissue using either internally or externally controlled optical contrast. In particular, diffuse optical tomography (DOT) is a technology that can provide tomographic images associated with the diffusion medium in the presence of scattering and absorption in the diffusion medium. For example, diffuse optical tomography (DOT) has been applied to blood flow dynamics imaging and chest tissue imaging of the brain. Typical diffuse optical tomography (DOT) methods and systems are described, for example, in International Patent Application No. PCT / US04 / 03229, entitled "Free Space Optics of Dispersion Medium," filed on February 5, 2004 by Vasilis Ntziachristos and Jorge Ripoll. Methods and systems for tomography.

Because of the low tissue absorption in the so-called near-infrared window, light with a wavelength in the near-infrared region has been known to propagate through the tissue for a distance of several centimeters. Near-infrared (NIR) windows enable the development of near-infrared (NIR) fluorescence technology that visualizes certain biochemical events inside living specimens.

Various methods are also being developed that relate to processing near infrared (NIR) fluorescence signals. The development of particularly suitable imaging systems has enabled the application of fluorescent molecular tomography (FMT), a technique for resolving molecular signatures in deep tissues using near-infrared (NIR) fluorescence probes or markers. Fluorescent molecular tomography (FTM), which is used to three-dimensionally image the activity of enzymes in cancers deep in vivo, is being tested.

A common assumption in conventional near-infrared (NIR) optical tomography is that propagation in diffusion media has high scattering but relatively low absorption, as is the case with near-infrared (NIR) windows. This assumption is based on the diffusion equations associated with the transport equations, using diffuse approximation, which provides an effective means for modeling near-infrared (NIR) photon propagation in tissues. Enable induction Such transfer equations are described, for example, in K.M.Case and P.F.Zweifel's "Linear Transport Theory" and Phys. Rev. E. 50, 3634 (1994), described in K.Frurtsu and Y. Yamada's "Diffusion Approximation for Scattered Random Media and Its Application".

As is known, recently available fluorescent proteins utilize excitation light having a wavelength in the visible region. In addition, conventional fluorescent proteins emit fluorescent light when excited. Tomography images using visible light are complicated by the relatively high absorption of visible light propagating in the tissues of living organisms, as is the case with conventional fluorescent proteins, which shows a clear attenuation. Due to the high absorption (eg in the case of visible light) the conventional diffusion approximation described above is not valid.

Other more advanced solutions (except the diffusion approximation described above) to the transfer equation have been made and are being applied to near infrared (NIR) optical tomography. The advanced solution overcomes the inadequacy of the diffusion approximation mentioned above. However, advanced solutions to the transfer equations are generally impractical because they are computationally complex and result in large data sets for tomography systems with large numbers of excitation light sources.

Many conventional optical tomography systems use optical switches as part of a light source assembly that is used to project a single light element at various angles or locations with respect to the specimen in order to provide multiple images necessary for tomography. Optical switches have been known to cause energy losses. In addition, many optical tomography systems use CCD cameras to collect light at room temperature or at moderate cooling. CCD cameras at room temperature or properly cooled show a relatively high degree of dark (thermal) noise, which has been known to tend to limit the quality of optical tomographic images obtained.

According to one embodiment of the invention, an optical tomography system includes an apparent light source modified to project excitation light towards a sample having a fluorescent protein therein. The excitation light enters the sample and becomes internal light in the sample. The internal light is modified to excite fluorescence from the fluorescent protein. The internal light and the fluorescence are diffused. In another embodiment of the present invention, at least one of the excitation light and the fluorescence has a wavelength in the visible light region.

According to another embodiment of the present invention, the optical tomography method includes generating the excitation light with an apparent light source modified to project the excitation light toward a sample having a fluorescent protein therein. The excitation light enters the sample and becomes internal light in the sample. The internal light is modified to excite fluorescence from the fluorescent protein. The internal light and the fluorescence are diffused. In another embodiment of the present invention, at least one of the excitation light and the fluorescence has a wavelength in the visible light region.

According to yet another embodiment of the present invention, the optical tomography system includes at least one member that is selectively movable to selectively move a clear light source that directs a plurality of light paths toward the specimen.

Before describing the imaging method and system according to the present invention, some preliminary concepts and terms will be explained. As used herein, "phantom" refers to a control subject to be imaged. The phantom is a typical article of manufacture with diffused light transmission characteristics similar to living tissue, for example plastic pieces. In another example, the phantom may be a vial having cells expressing a fluorescent protein, such as a fluorescent marker.

The term "apparent light sources" as used herein is used to describe the projection of a single light source to multiple physical positions or angles, each providing a distinct light source.

As used herein, “excitation” light is used to describe light generated by an excitation light source (eg, a clear light source), and refers to light before going to a sample to be imaged and entering the sample. In this case, once in the sample, the light is referred to as "intrinsic" light. The internal light is absorbed into the sample and exits the sample as scattered light.

The internal light exiting the sample is generated by the excitation light source and has the same wavelength as the excitation light. The excitation light and internal light may be monochromatic or include a broader spectrum, such as white light.

In one embodiment of the invention, the internal light exiting the sample is received by a light detection device that is generally on the same side with respect to the sample as a light source (eg, in the reflection image of FIG. 1B to be described below). In another embodiment of the invention, the internal light exiting the sample may be received by a light detector (eg, in a transmission illumination image to be described in FIG. 1A below) that is generally placed opposite the excitation light source for the sample. have. In addition, the excitation light becomes internal light when the excitation light enters the sample, is reflected in or passes through the sample.

The term "emitted" light, as used herein, is intended to describe light generated in or by biological tissue. As used herein, the terms "fluorescence" or "fluorescent" are used to describe the form of emitted light resulting from excitation of a fluorescent protein in response to internal light.

As used herein, "image" is used to describe a visual representation with basic "image data" generated by a digital camera or computer system. However, the term "image" may also be used to refer to image data as used herein.

As used herein, "diffuse" is used to describe light that has photons that face several scatterings (eg, ten or more times) when propagated in the sample without absorption of photons in the sample. The number of such scatterings may be 10 or more times or less.

The method and system of the present invention are described below for application to visible light propagating in biological tissues where diffuse light propagation is dominant. However, the above methods and systems are equally well applicable to any form of light propagating in any medium in which diffusion propagation prevails, e.g. excitation visible light and near infrared (NIR) fluorescence propagating at sufficiently deep distances in biological tissues. It can also be applied to near infrared (NIR) light propagation such as (radiation). The method and system may also be applied to light propagating in a medium in which interference propagation is dominant.

Although the method and system according to the present invention have been described as applying fluorescent proteins that emit visible fluorescence because they provide particular benefits in the visible wavelength range of about 400 nm to 700 nm, the methods and systems have different wavelengths. Light, for example, fluorescence in the near infrared (NIR) region of about 700 nm to 1000 nm may also be applied. In addition, the method and system according to the present invention allow the excitation light to be in one wavelength region, for example in the visible region, and the fluorescence emitted by the fluorescent protein in another wavelength region, for example in the near infrared (NIR) region. The same applies to existing systems. The method and system according to the invention can also be applied when the fluorescence emitted by the excitation light and the fluorescent protein is both in the near infrared (NIR) region or both in the visible region. In addition, light beyond the wavelength range of about 400 nm to 1000 nm may also be used.

The above-described features of the present invention will be more clearly understood by the detailed description of the drawings as well as the invention itself.

FIG. 1A is a block diagram of an optical tomography system capable of providing a transillumination image.

1B is a block diagram illustrating an optical tomography system capable of providing a reflected image.

2 is a block diagram showing an optical tomography system having a laser source, an optical fiber, an optical switch, a cooled CCD camera and an image processing apparatus.

3 is a block diagram of an optical scanner with fiber connectors, position control mirrors and telecentric lenses used together for the generation of a number of distinct light sources having a flat light field at a constant working distance WD.

4 is a flowchart illustrating a processing process used to provide a tomography image according to an embodiment of the present invention.

FIG. 5A shows 31 series of images corresponding to the 31 clear light sources of FIG. 1A provided by the optical scanner of FIG. 3, showing internal light (excited light entering and exiting the sample from the clear light source). .

FIG. 5B shows 31 different images corresponding to the 31 distinct light sources of FIG. 1A provided by the optical scanner of FIG. 3, which shows the fluorescence emitted from the fluorescent protein in the sample in response to the internal light of FIG. 5A.

FIG. 5C is provided by the optical scanner of FIG. 3, showing another 31 images corresponding to the 31 clear light sources of FIG. 1A, which do not pass through the sample but have a uniform slab (ie, phantom). Show the transmitted light passing through.

FIG. 6A shows a fluorescence image superimposed on a white light image of a dead rat with a vial with green fluorescent protein expressing cells located in the esophagus.

FIG. 6B shows a fluorescence image superimposed on a white light image of a dead rat with another vial with green fluorescent protein expressing cells located in the esophagus, wherein the vial has a greater number of cells than the bottle of FIG. 6A. have.

7A is a block diagram of a system of optical tomography with a planar imaging chamber.

FIG. 7B is a block diagram illustrating an imaging chamber used in the system of FIG. 7A.

8A is a block diagram showing an optical tomography replacement system with a rotating planar imaging chamber.

FIG. 8B is a block diagram of a rotating planar imaging chamber that may be used in the system of FIG. 8A.

9 is a block diagram showing another alternative system of optical tomography with a rotating cylindrical imaging chamber.

FIG. 10A is a block diagram showing another alternative system of an optical imaging device having a rotating cylindrical imaging chamber and an apparently movable light source.

10B is a block diagram illustrating a rotating cylindrical imaging chamber and multiple light sources.

FIG. 11A is a block diagram showing another alternative system of an optical tomography camera with an optical scanning head having a clear array of light sources.

FIG. 11B is a block diagram of the optical scanning head shown in FIG. 11A.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited to the following embodiments, and those skilled in the art will understand the technical spirit of the present invention. The present invention may be embodied in various other forms without departing from the scope thereof.

Referring to FIG. 1A, an optical imaging system 10 using fluorescent proteins includes an imaging light source 12 and a light directing device that provide a number of distinct light sources (not shown). (14). The apparent light source provides excitation light 22a to 22c at various locations with respect to the specimen 18. Although three positions are shown, the apparent light source may be more or less than three. The excitation light 22a, 22c strikes the sample 18 and enters the sample 18 to become the internal lights 24a and 24b, and these internal lights 24a and 24b exit the sample 18. The internal light 24a, 24b is optionally received by the photo detector 32 through the selection light filter 28, the image intensifier 30. The excitation light 22b also enters the sample 18 and strikes the fluorescent protein 20 within the sample 18. In response to the excitation light 22b, the fluorescent protein 20 emits fluorescence 26, and the fluorescence 26 optionally passes through the selection light filter 28 and the image intensifier 30 to the photodetector 32. Are accepted.

Optionally, the white light source 40 may further provide illumination of the sample while providing another optical path (not shown), wherein the white light is reflected from the surface of the sample and optionally the optional light filter 28 and image enhancer ( 30 is received and received in the photo detector 32.

In one embodiment of the invention, the internal light 24a, 24b, the fluorescence 26 and the white light from the white light source 40 are simultaneously received by the photo detector. In such a device the internal light 24a, 24b, the fluorescence 26 and the white light from the white light source 40 are optionally separated by the selection filter 28 and provide different light to the photo detector 32 simultaneously or separately. . For this reason, the pass band of the selection filter may be concentrated in different time zones depending on the wavelength of the internal light, the fluorescence and the white light.

In another embodiment of the present invention, one or more of the internal light 24a, 24b, the fluorescence 26 and the white light from the white light source 40 are combined with the internal light 24a, 24b, the fluorescence 26 and the like. The white light from the white light source 40 is received at a different time zone than the other lights. For example, internal light 24a, 24b is first received, and image source 12 is extinguished. Fluorescence 26 is received after the interior light 24a, 24b is no longer present. After the fluorescence 26 stops emitting, the white light source 40 is turned on and the white light is received.

The photo detector 32 converts the received light into digital data 32a (also referred to herein as image data). The image processing device 34 receives digital data 32a and produces an image 46. In another embodiment of the invention, the image 46 is a tomography image.

The image processing apparatus 34 may include a transmission problem processing apparatus (FP) 36 having a diffusion equation processing apparatus 38. The functions of the transmission problem processing apparatus 36 and the spreading equation processing apparatus 38 are explained fully below with reference to FIG. 4, for example. It is sufficient here to say that the transmission problem processing apparatus 36 compares the light propagation (excitation light) in the specimen with the light received in the photo detector. The difference between the received light and the expected light (provided by the light propagation model) is related to the fluorescent protein 20 in the sample. The diffusion equation processing apparatus 38 uses a modified diffusion coefficient, which is used in the "diffusion equation" associated with the "transmission equation" that can be used to provide the light propagation described above.

In some embodiments of the present invention, the modified diffusion coefficients enable the model to predict light propagation for light in the visible wavelength range having a wavelength on the order of about 400 nm to about 700 nm. In another embodiment of the present invention, the modified diffusion coefficients enable the model to predict light propagation for light in the near infrared wavelength range having a wavelength on the order of about 700 nm to about 1000 nm. In another embodiment of the present invention, the modified diffusion coefficient allows the model to predict light propagation of light having a wavelength outside the region of about 400 nm to 1000 nm.

System 10 may also include a light direction controller 44 that directs the apparent light source to an already determined light path. The system 10 is provided with an imaging chamber 16 along with the position of the light direction controller 44 or the light direction controller to provide a clearer light source, such as internal light passing through the sample 18 along a predetermined light path. It may optionally include a chamber position controller 42 that can be used to move.

It is to be understood that system 10 can provide a transmissive illumination imaging system in which light generated by imaging light source 12 passes through sample 18 and is essentially received on the other side of sample 18.

Referring to FIG. 1B, the system 70 uses the same reference numerals for the same members as those of FIG. 1A, such that the selection filter 28, the image intensifier 30 and the photo detector 32 are generally imaged. Like the light source 12 and the light indicating device 14 is located on the same side as the specimen 18. In this particular arrangement the interior light 72a, 72b is received in the photo detector 32 as reflected light. Inevitably, the excitation light 22a to 22c enters the sample 18 and is reflected or, in special cases, is scattered to the light receiver 32. Fluorescence 74 is also received by photodetector 32. System 70 generates image 76.

It will be appreciated that the system 70 can provide a reflective imaging system in which light emitted by the imaging light source 12 passes through the sample 18 and is essentially received on the same side as the sample 18. In another embodiment of the present invention, the angle between the light indicating device 14 and the light detector 32 is about 90 degrees.

2, the apparatus 100 includes a laser 102, an optical fiber 104, and an optical switch 106, which combine to have an imaging chamber having an imaging plate 108 on which the specimen 110 is located. Generate a number of distinct light sources that are directed towards 112). Imaging chamber 112 is filled with a matching fluid 114. The selection filter 115 and the CCD camera 116 receive the internal light passing through the sample 110 and the fluorescence emitted by the sample 110. As described above, the selection filter has a passband selectively concentrated at one wavelength of internal light or fluorescence depending on whether an internal image or a fluorescence image is generated. The optical switch 106 can be controlled by the computer 122. Computer 122 may also control CCD camera 116 via CCD controller 118. The image processing apparatus 120 receives the digital data 116a through the CCD controller 118. Graphical display 124 may show the resulting image information.

The laser 102 corresponds to the image light source 12 of FIGS. 1A and 1B, the optical switch 106 corresponds to the light indicating device 14 of FIGS. 1A and 1B, and the image chamber 112 is of FIG. 1A. And the CCD camera 116 corresponds to the photo detector 32 of FIGS. 1A and 1B, and the image processing apparatus 120 may correspond to the image processing apparatus of FIGS. 1A and 1B. 34).

The specimen 110 is represented here as a rat located in the imaging chamber 112. The laser 102 provides excitation light (not shown) that enters the sample 110 and excites (not shown) the fluorescent protein in the sample 110 to generate fluorescence (not shown). The CCD camera 116 receives laser light as internal light (which passes through the sample 110) and also receives fluorescence emitted from within the sample 110 through the selection filter 115.

In a particular embodiment of the invention, the laser 102 is an argon (Ar +) laser that emits laser light at about 200 Hz continuous wave (CW) power having a wavelength on the order of approximately 448 nm. The laser light is used to excite a green or yellow fluorescent protein, for example, in the sample 110.

In one embodiment of the present invention, the optical fiber 104 is a multi mode optical fiber having a diameter of about 100 μm. The laser light 102 provides a number of distinct light sources at different body positions relative to the specimen 110 in the manner of an optical switch 106. In another particular embodiment of the invention, the optical switch provides about 31 apparent light sources. However, in another embodiment of the present invention, more or less than 31 distinct light sources can be provided.

Although an optical switch 106 is shown, according to another embodiment of the present invention, the optical switch 106 may be replaced with an optical scanning head (or an optical scanner) shown in detail in FIG. 3.

The optical switch 106 may provide a clear light source at various angles or positions with respect to the sample 110, whereby the image processing apparatus 120 may form various corresponding images, and the various images may be image processing apparatuses. 120 may be combined in the tomography process.

The optical switch 106 includes a plurality of optional optical fiber paths (not shown) that are modified to direct light to the corresponding plurality of selective fixed body positions, the optical switch being clearly fixed selectively in position and number. Provide a light source.

In one embodiment of the invention, the living tissue, represented here as a rat, is placed on the optical imaging plate 108 in contact with the optical matching fluid 114. Matching fluid 114 is further described below. Matching fluids are used to reduce the effects of stray light. However, other embodiments of the present invention may not use a matching fluid.

In operation, the internal light (which originates from each of the apparent light sources) and also the light emitted by the fluorescent protein in the sample 110 is received by a CCD camera, for example as described in connection with FIG. 4, and then the image. It is processed for tomography imaging by the processing apparatus 120.

In a particular embodiment of the present invention, the CCD camera 116 is a cooled CCD camera with reduced dark noise. For example, the CCD camera 116 may be a Princeton Instruments CCD camera from Roper Scientific, having a cryogenic cooling unit.

During operation, the optical switch 106 is controlled and operated by the CCD controller 118 so that each obtained image corresponds to a new position of a new clear light source (i.e., a different path in the light switch 106). Proper synchronization of excitation and detection can be achieved. Each capture consists of N images, one capture for each distinct light source location. Therefore, assuming a 512 x 512 pixel CCD camera, the maximum number of data for each measurement set is N x 512 x 512. However, the number of detectors (e.g. pixels) used for continuous processing by the image processing apparatus 120 may be less than the entire group of 512 x 512 pixels, which is a field of view associated with each of the light sources where the number of detectors is apparent. of view). The number of pixels used can also be reduced to reduce the computation time required for the image processing device.

In one embodiment of the present invention, the CCD camera 116 and the selection filter 115 have a sample in the direction indicated by arrows 140, 142, for example, to obtain more images at different angles relative to the sample 110. May optionally move relative to 110. However, in another embodiment of the present invention, the optical scanner 117, which is somewhat remote from the CCD camera 116, may provide an image of a different angle with respect to the sample 110.

The specimen 110 may be placed horizontally on the image plate 108 and pressed into a cover glass (not shown). The imaging chamber 112 is filled with a matching fluid 114. According to one embodiment of the present invention, the matching fluid 114 is composed of intralipid and Indian ink solution. Matching fluid 114 provides a match of optical properties, which tends to reduce refractive index and diffuse wave mismatches in the chamber. In one embodiment of the invention, the matching fluid 114 consists of about 1% of intralipid and about 2.1% of ink, μ _a = 1.25 cm ⁻¹ , μ _s `= Corresponds to 1.67 cm <sup>−1</sup> , where each μ <sub>a</sub> is an absorption coefficient and μ <sub>s</sub> `is a reduced scattering coefficient. The reduced scattering coefficient is described below.

Referring again to FIG. 3, the optical scanner 150 may be used at the position of the optical switch 106 of FIG. 2, and may also be used as the optical scanner 117 of FIG. 2. In one embodiment of the present invention, the optical scanner 150 generally comprises two galvanometer controlled mirrors (shown here for clarity), which rotate about a vertical axis. It includes a scanning lens 158 that collects and scans the light beam 156 into the input window of the imaging chamber 162 to provide a number of distinct light sources at each different body location represented by light rays 160a and 160b. In one embodiment of the invention, the scanning lens 158 is a telecentric lens. In one embodiment of the present invention, the light beam diameter in the field plane (eg, the imaging chamber 112 of FIG. 2) is approximately 300 μm. A single light source (not shown) may be used, which is received by the fiber connector 152.

The optical scanner 150 has only a small light loss compared to the light loss of a conventional optical switch such as, for example, the optical switch 106 of FIG. Therefore, the optical scanner 150 can provide a lower loss of apparent light source system. In addition, in the case of the optical scanner 150, unlike the optical switch 106 shown in Fig. 2 in which a fixed number of light paths are in a fixed position, the number of light sources and the positions of the light sources are clear as well as the scanning area and the light beam The shape of the can vary significantly. It is also possible to achieve higher light voltages and wider light wavelength ranges (near infrared in the visible).

Optical scanner 150 has many advantages over optical switches, including but not limited to lower energy loss, uniform response to many apparent light sources, and improved reliability and intensity. In addition, the number of spatial sources and the spatial shape as well as the scanning area can be controlled by software, thus varying according to the characteristics of the sample being scanned. It is also possible to achieve higher voltage illumination and wider wavelength ranges (eg, on the order of 400 nm to 1000 nm).

Referring to FIG. 4, the method of using the optical tomography apparatus 200 starts at step 202 of transmitting light, in which excitation light is generated to pass through a sample. In other embodiments, for example, in the case of tomography imaging, the excitation light is provided by a number of distinct light sources corresponding to different locations on the sample.

In another embodiment of the present invention, a plurality of light sources generally provide excitation light at a number of distinct light sources. According to another embodiment of the invention, a number of distinct light sources in turn provide excitation light.

In step 204 of receiving light, the internal light is received. As described above, the internal light also corresponds to excitation light exiting through the sample. The internal light may be received in a light detector such as, for example, the photo detector 32 of FIG. 1A. In another embodiment of the invention, the received internal light corresponds to transmitted illumination light, in which case the light detector and the apparent light source are necessarily on the opposite side of the sample. In another embodiment of the invention, the received internal light corresponds to reflected light, in which case the photo detector and the apparent light source necessarily lie on the same side of the specimen.

In the fluorescence receiving step 206, fluorescence is received. The fluorescence is emitted by fluorescent protein in the sample in response to the internal light. The fluorescence is received, for example, in a photo detector such as photo detector 32 of FIG. 1A. In another embodiment of the present invention, the fluorescence and the internal light can be simultaneously received by the method of the optical filter modified to separate the internal light and the fluorescence (for example, reference numeral 28 of FIG. 1A). In another embodiment of the present invention, the fluorescence may be received by an optical filter method after the excitation light is extinguished.

In step 208 of converting the first image information, the received light is converted into first image information such as the digital data 32a of FIG. 1A. In operation 210 of converting to second image information, the received fluorescence may be converted into second image information, which may also be digital data 32a.

In the model generation step 212, a model is generated to predict light propagation in a specimen. The model is based on, for example, a diffusion equation having a modified diffusion coefficient fully described in Equation 4 below and a modified wave number according to Equation 6 below.

The optical model generated in model generation step 212 is related to propagation in a uniform medium, ie a medium in which no optical heterogeneous component is present. In other embodiments of the present invention, further refined models may be used to solve and solve information regarding background optical heterogeneity.

Light propagation in tissues can be modeled using diffusion equations with modified diffusion coefficients, where the modified diffusion coefficients have relatively high absorption of the medium, for example in the case of visible light propagation in biological tissues. It will be evident as described below to be modified to anticipate the characteristics of light propagation to the diffusion medium. For example, a modified diffusion equation with modified diffusion coefficients, which will be described below, for the propagation of visible light that is known to diffuse in biological tissue and has a relatively high absorption can be expected. However, the modified diffusion equation is also suitable for accurately predicting the propagation of light with other wavelengths transmitted in the diffusion medium, for example near-infrared propagation in biological tissues.

의 형광 단백질로 인해 매질 내에서 발생한 형광 필드(field)를 독립적으로 계산하고, 그 후에 노말라이즈(normalize) 된 Born 필드(확산 근사)를 계산하기 위해 사용한다. 다음에서 상세하게 설명하는 바와 같이 수정된 확산 방정식은 매질 내부의 형광 단백질의 영상을 제공하는 전송 문제(forward problem)에 사용될 수 있다.The general diffusion equation can be derived from the Radiative Transport equation. Internal light fields and locations generated by laser excitation light (internal light) propagation in the medium

The fluorescence field generated in the medium due to the fluorescent protein of is independently calculated and then used to calculate the normalized Born field (diffusion approximation). As described in detail below, the modified diffusion equation can be used for a forward problem that provides an image of the fluorescent protein inside the medium.

에서의 분명한 광원으로 인한 광 검출기 위치

에서 검출된 광 전파에 대한 매질에서의 Born 필드는

다음과 같이 주어진다.Positions with both modified diffusion coefficients and modified wave propagation to account for high absorption

Photodetector location due to the clear light source at

The Born field in the medium for the light propagation detected at

Is given by

[Equation 1]

(

,

)=

(

,

) =

(

,

)와

는 각각 여기

(레이저)와 방사

(형광) 파장이고

는 신호로 인한 번짐이고,

는 예를 들면 도 1a의 선택 필터(28)와 관련된 대역 통과 필터 감쇄 인자이고,

는 여기

와 방사

파장에서 기기 이득 차이(instrument gain difference)(예를 들면, 광 검출기 이득 차이)를 설명하는 이득 항이며,

은 형광 단백질 흡수 계수와 형광 양자 생산의 결과이고,

은

과

각각에서 높은 흡수성을 설명하는 수정된 광자 파 전파 파수이며,

은 매질에서 광의 속도이고,

는

에서 높은 흡수성의 존재 하에 수정된 확산 계수이고

은

의 매질에서 위치

에서 만들어진 광자 필드를 설명하는 항이다. 그리고

는 광 검출기에 대한 위치

에서 형광 단백질로부터의 방사 광자파의 전파를 설명하는 확산 근사에 대한 그린 함수 답(Green`s function solution)이다. 함수

는 다음 수학식 2와 같다.In Equation 1,

(

,

)Wow

Are each here

(Laser) and spinning

(Fluorescence) wavelength

Is the bleeding from the signal,

Is the band pass filter attenuation factor associated with the selection filter 28 of FIG.

Is here

And spinning

A gain term that describes the instrument gain difference (eg, photodetector gain difference) at wavelength,

Is the result of fluorescence protein absorption coefficient and fluorescence quantum production,

silver

and

Is a modified photon wave propagation wave that explains the high absorption at each,

Is the speed of light in the medium,

Is

Is a modified diffusion coefficient in the presence of high absorption at

silver

Position in the medium

This section describes the photon field created by. And

Position for photo detector

The Green's function solution to the diffusion approximation describing the propagation of radiated photon waves from fluorescent proteins in. function

Is as shown in Equation 2 below.

[Equation 2]

에 의해 노말라이즈된다. 상기 수학식 1에서 노말라이제이션 사용의 장점은 위치 의존적인 기여가 제거되고 또한 이 필드가 형광 단백질의 존재 하에서도 계산이 가능하다는 것이다. 이것은 형광 단백질의 투여 전에 백그라운드의 측정이 필수적이지 않다는 것을 의미하며, 생체 연구에서는 중요하다.Equation 1 is essentially

It is normalized by. An advantage of using normalization in Equation 1 is that position dependent contributions are eliminated and this field can be calculated even in the presence of fluorescent proteins. This means that background measurements are not essential prior to administration of the fluorescent protein, which is important for in vivo studies.

A useful way of expressing the absorption dependence of diffusive light propagation independent of medium absorption is by using a diffusion coefficient as in Equation 3 below.

[Equation 3]

는 일반적으로 매질의 흡수, 산란 및 이방성에 의존하는 상수이다. 계수

는 감소된 산란계수이고

는 흡수 계수이다. 감소된

는

로 쓸 수 있고, 여기서

는 산란 계수이다. 높은 흡수성을 설명하는 수정된 확산 계수

에 대한 표현을 Radiative Transport 방정식으로부터 유도를 통해 다음 수학식 4와 같이 얻을 수 있다는 것을 발견할 수 있다.In Equation 3,

Is a constant that generally depends on the absorption, scattering and anisotropy of the medium. Coefficient

Is the reduced scattering coefficient

Is the absorption coefficient. Reduced

Is

Can be written as

Is the scattering coefficient. Modified Diffusion Coefficient Demonstrating High Absorption

It can be found that the expression for can be obtained as shown in Equation 4 through derivation from the Radiative Transport equation.

[Equation 4]

는 이방성 인자이다. 여기서

는 감소된 산란 계수

의 항으로 표현되는데, 이것은 이방성 매질에서 산란 실험에 적절한 양이다. 수학식 4와 대부분의 표준적인 확산 계수 D에 대해 흔히 가장 흔하게 사용되는 표현 사이의 하나의 주요한 차이는 흔히 사용되는 표현에서

의 값이

또는

로 원칙적으로 고정된다는 것이다. 그러나 고려되는 스펙트럼 영역에 의존하기 때문에

에 대한 더 일반적인 표현은 이방성 인자

의 적절한 선택에 의해 백그라운드 흡수 정도의 다양화를 설명할 수 있다. 적절한 값은 분석적 또는 실험적으로 찾을 수 있다.

에 대한 표현은 상기 수학식 4 및 수학식 3에서 다음 수학식 5와 같은 형태로 찾아진다.In Equation 4,

Is an anisotropic factor. here

Is the reduced scattering coefficient

It is expressed in terms of, which is an appropriate amount for scattering experiments in anisotropic media. One major difference between Equation 4 and the most commonly used expressions for most standard diffusion coefficients D is in the commonly used expressions.

Has a value of

or

In principle, it is fixed. But because it depends on the spectral region under consideration

More general expression for anisotropic factor

By varying the degree of background absorption can be explained by appropriate selection. Appropriate values can be found analytically or experimentally.

The expression for is found in Equation 4 and Equation 3 as shown in Equation 5 below.

[Equation 5]

에 대한 전형적인 값은 약 0.2 내지 0.6 정도이다. 생물학적 조직에서 가시광선 전파의 경우, 이방성 인자

를 0.8이라고 가정하면,

는 약 0.50 내지 0.55 정도이다.

에 대한

의 의존성은 작고 실제의 생물학적 값(

는 0.5 내지 0.9사이)안에서

의 값의 변화는

값에 작은 변화를 준다.In Equation 5,

Typical values for are about 0.2 to 0.6. Anisotropic Factors for Visible Light Propagation in Biological Tissues

Assuming 0.8

Is about 0.50 to 0.55.

For

The dependence of is small and the actual biological value (

Is between 0.5 and 0.9)

The change in the value of

Make a small change in the value.

또는

) 확산 근사는 높은 흡수성을 가진 매질에 대하여 부정확한 결과를 생산한다는 것을 이해할 수 있을 것이다. 이것이 높은 흡수성의 존재(예를 들면, 생물학적 조직 내에서 가시광선의 전파)하에서 확산 근사가 실패하는 이유라고 오랫동안 생각되어 왔다. 그러나 상기 수학식 4의 수정된 확산 계수가 사용되면, 확산 근사는 정확해진다. 그런 이유 때 문에, 수정된 파수는 다음 수학식 6과 같이 정의되어야 한다.If existing coefficients are used (i.e.

or

It will be appreciated that diffusion approximation produces inaccurate results for media with high absorption. It has long been thought that this is the reason why diffusion approximation fails in the presence of high absorption (eg, propagation of visible light in biological tissue). However, if the modified diffusion coefficient of Equation 4 is used, the diffusion approximation is accurate. For that reason, the modified wave number should be defined as in Equation 6 below.

[Equation 6]

는 변조 주파수(연속 파 여기 광의 경우

=0)이다.In Equation 6,

Is the modulation frequency (for continuous wave excitation light

= 0).

Combining the wave propagation wave number of Equation 6 and the modified diffusion coefficient of Equation 4 provides a green function answer for diffusion approximation suitable for imaging in the presence of high absorptivity such as propagation of visible light in biological tissue. This can be induced.

The above described models (descriptive equations followed by Equation 1) can take advantage of apparent light source properties such as their position or intensity, for example.

In step 214 of combining the first image information, the second image information and the model, the first image information, the second image information and the light propagation model are combined, for example in a so-called transmission model. When the sample is not internally uniform as it has an internal fluorescent protein, the binding in step 214 of combining the first image information, the second image information and the model may be in the form of an "image problem" or "transport model". Generates. I.e. measurement = (theoretical prediction) X (unknown distribution), where the measurement is switched from the step 204 to receiving the second image information by means of the photo detector 32 (see FIG. 1A). The theoretical predictions generated in step 210 are generally provided in the model generation step 212 by the image processing apparatus 34 (see FIG. 1A) in accordance with Equations 1-6. This unknown distribution corresponds to the fluorescence emitted by the fluorescent protein. The image processing device 34 may also solve for unknown distributions to set the features and body position of the fluorescent protein 20 (see FIG. 1A) in the sample 18 (see FIG. 1A).

In step 216, generating a concentration map of the fluorescent protein, a map is generated of the concentration of fluorescent protein in the sample. In one embodiment of the invention, the map is a tomography image. In order to generate such a map, the described transport model is "inverted" to solve the unknown distribution described above.

In the step 216 of generating the fluorescent protein map, the target volume is divided into vertical (horizontal) layers (for example 21 layers) each containing a large number of voxels (for example 651). do. The size of the voxel is selected based on the size of the view field and the number of fractions.

The volume of interest can be fractionated into many voxels in three dimensions. This may appear to cross horizontally, vertically, or layers, because it resembles cuboids stacked next to each other in three dimensions. Each of the voxels has an unknown amount of fluorescent protein and an unknown attenuation. If fluorescence and attenuation are known in each voxel, the measured image can be expected. However, fluorescence and attenuation in each voxel are unknown. Therefore, the inverse may be taken to solve the above described transmission model with respect to the above described unknown distribution in order to create a map.

In another embodiment of the present invention, overlapping a white light image of a sample onto a map of the fluorescent protein concentration may be desirable for enhanced understanding of the image. For this reason, the white light can be illuminated in the step 218 of transmitting the white light to the sample, the white light is received in the receiving 220 of the reflected white light, and the white light image is generated in the step 222 of generating the white light image information. Can be generated.

From the step 216 of generating the map of the fluorescent protein concentration in the step 224 of superimposing the white light image information on the fluorescent protein map the white light image generated in the step 222 generating genital white light image information It can be superimposed onto the resulting fluorescent protein map. For this reason, in one embodiment of the present invention, the white light image may be recorded or arranged together with the map of the fluorescent protein.

In order to arrange the white light image on the map of the fluorescent protein, for example, a clear light source may be determined by passing through a phantom such as a clear light source image. This process improves the simultaneous recording of white light images that can be superimposed on tomographic images while reducing relative position errors.

Referring to FIG. 5A, 31 images 300 of internal light correspond to 31 distinct light sources that direct light to the specimen and internal light received therefrom as transmitted illumination light. Obvious light sources direct light to the specimen one by one or simultaneously or in combination.

Image 300 may occur using a bandpass interference filter (eg, reference 28 in FIG. 1A) concentrated at the wavelength of excitation light from a light source (eg, reference 12 in FIG. 1A).

Referring to FIG. 5B, 31 images 310 of fluorescent proteins correspond to 31 distinct light sources that direct light to the sample and radiated fluorescence received therefrom. The light sources are directed one by one or simultaneously or in combination to direct the light to the specimen. Fluorescence can be received at the same time directing a clear light source towards the sample or after directing a clear light source towards the sample. The internal optical image of FIG. 5A and the fluorescent image of FIG. 5B are combined, for example, in step 214 of combining the first image information, the second image information, and the model of FIG. 4.

Image 310 may be generated using a band pass interference filter (eg, reference 28 in FIG. 1A) concentrated at a wavelength of light emitted from a fluorescent protein (eg, reference 20 in FIG. 1A).

를 발생시키는데 사용할 수 있다. 역동적인 영역을 최대화하기 위하여 노출된 시간을 각 영상마다 다양화 할 수 있다.Measurement (or image) 300, 310 field described by Equation 1 above

Can be used to generate The exposure time can be varied for each image to maximize the dynamic range.

Referring to FIG. 5C, the 31 images 320 of the apparent light source correspond to the 31 apparent light sources that direct light to pass through the phantom and the light received therefrom. Obvious light sources direct light one by one or simultaneously or in combination with the phantom. An image of the apparent light source may optionally be used to record the white light image with the concentration map of the fluorescent protein, for example as shown in step 224 of superimposing the white light image information on the fluorescent protein map of FIG. 4.

Image 320 may be generated using a band pass interference filter (eg, reference 28 in FIG. 1A) concentrated at the wavelength of excitation light from a white light source (eg, reference numeral 40 in FIG. 1A).

6A and 6B, examples of images provided by the method and system according to the present invention can be seen. Another number of green fluorescent proteins (GPFPs) expressing cancer cells, namely about 10 ⁵ and about 10 ⁶ , were used. Fluorescent proteins were placed inside thin glass tubes and mice were killed and inserted into the esophagus of the animals. The animal is placed inside an imaging chamber (eg, reference numeral 16 in FIG. 1A) and the imaging chamber is filled with the matching fluid described above. Imaging is done using an array of 31 distinct light sources that transmit light through the area around the chest of the animal.

First, referring to FIG. 6A, the image 350 includes a map 352 of about 10 ⁵ fluorescent proteins in which a white light image 354 of a mouse is superimposed on the map.

Next, referring to FIG. 6B, the image 370 includes a map 372 of about 10 ⁶ fluorescent proteins in which a white light image 374 of a mouse is superimposed on the map.

Referring to FIG. 7A, the system 400 for the optical tomography 400 includes a laser source 402 coupled with the optical scanner 412. The laser source 402 provides a number of distinct light sources, for example as described in connection with FIG. 3. System 400 also includes a laser intensity controller (LIC) 406. Laser intensity controller (LIC) 406 provides intensity control for laser source 402.

System 400 further includes an image intensifier 420 and a CCD camera. The selective light filter 418 is selectively band focused to the wavelength of the light emitted by the fluorescent protein in the sample 416 or the wavelength of the excitation light transmitted by the apparent light source. Computer 424 controls laser intensity controller 406 and optical scanner 412. The computer 424 also functions as an image processing device, for example as the image processing device 34 of FIG. 1A, which provides an image on the graphic display 426.

In operation, the optical scanner 412 provides a number of distinct light sources at a corresponding number of locations relative to the specimen 416. The CCD camera collects the internal light passing through the sample 416 as transmitted illumination light and also the fluorescence emitted by the fluorescent protein in the sample 416. The CCD camera converts the received light into digital data and the computer processes the data as described above with respect to FIG. 4 to provide tomographic images on the graphic display 416.

In one embodiment according to the invention, the plurality of images necessary for tomography imaging are related to the location of a number of distinct light sources provided by the optical scanner 412 and the imaging chamber remains sufficiently stationary but the quality of the images. It can move along the axis 417 to affect it.

7A and 7B, imaging chamber 414 includes a cylinder 450, which may be filled with the matching fluid described with reference to FIG. 2, for example. Imaging plate 454 and lid glass 456 surround sample 416.

Referring to Fig. 8A, the same reference numerals are used for the same members as those of Figs. 7A and 7B. System 500 for optical tomography includes an imaging chamber 504 holding the specimen 510. Imaging chamber 504 is deformed to rotate and move parallel along axis 508 as indicated by arrow 506. System 500 may include a rotating stage 502 that includes an imaging chamber 504.

As described in connection with FIG. 7A, the number of images necessary for tomography images relates to the location of a number of distinct light sources provided by the optical scanner 412. Imaging chamber 504 can also rotate and move in parallel to provide, for example, the position and / or angle of the light source more clearly under the control of computer 424 and provide a better image of sample 510.

Referring to FIG. 8B, the same reference numerals are used for members such as those of FIGS. 7A and 8A, wherein the imaging chamber 504 of FIG. 8A may be replaced with the imaging chamber 550 as shown in FIG. 7B, for example. However, it can be rotated as indicated by arrow 552 and deformed to be able to move in parallel as indicated by arrow 554.

The imaging chamber 550 can also move in parallel along the axis indicated by the arrow 556 and this movement takes into account that the image obtained by the CCD camera 442 of FIG. 8A is affected.

9, the same reference numerals are used for the same members as those of FIG. 7A. Another system 600 for an optical tomography system generally includes a cylindrical imaging chamber 602 for placing the specimen 604. Imaging chamber 602 may rotate as indicated by arrow 606. This rotation can be adjusted by the computer, for example, with a rotation stage controller 612 controlled by the computer 608. The laser intensity controller (LIC) 406 of FIG. 7A is included in the system 600 although not shown.

Many of the images necessary for the tomographic images, as described in connection with FIG. 7A, are related to the location of a number of distinct light sources provided by the optical scanner 412. In addition, the imaging chamber 602 is rotated to provide a better image of the specimen 604, for example, to provide a more apparent light source position and / or angle under computer control.

An advantage of the cylindrical imaging chamber 602 is that the rotation is fast and simple, as well as imaging operations, without deterioration of the image quality.

With reference to FIG. 10A, another system 700 for an optical tomography apparatus includes a laser source 710, which provides light to the optical fiber 712. Optical fiber 712 may be selectively moved along at least two axes 718, 720, for example by structure 716. System 700 may also generally include a cylindrical imaging chamber 722 for placing the specimen 724. Imaging chamber 722 may rotate as indicated by arrow 723. Rotation may be computer controlled using, for example, rotation stage controller 744 controlled by computer 740. The movement of the structure 716 which also provides the movement of the fiber 712 over at least two axes 718, 720 can be computer controlled by the XY-stage controller 746 controlled by the computer 740, for example. . The laser intensity controller (LIC) 406 of FIG. 7A is included in the system 700 although not shown.

As in the system described above, the internal light passing through the sample 724 and the fluorescence emitted within the sample 724 may pass through the selection light filter 734 and be received by the image intensifier 736 and the CCD camera 738. The CCD camera 738 provides the digital data 738a to the computer 740, which converts the data 738a to at least the first image information of FIG. 4 to generate a map of fluorescent protein concentration. The process shown in step 216 and a map of the fluorescent protein in the sample 724 on the graphical display 742 is provided.

The other optical fiber 714 can provide a second light source 726 opposite the sample 724 relative to the optical fiber 712. The second light source 726 may be a single white light used to provide a white light image of the step 218 of transmitting white light to the sample of FIG. 4 to superposing the white light image information on a map of fluorescent protein 224. . In another embodiment of the present invention, the second light source 726 is at least two axes 730 to provide a number of distinct light sources used in the reflected image of the specimen 724, for example, in the same manner as shown in FIG. 1B. , 732).

The same high quality three dimensional image provided by system 600 of FIG. 9 may also achieve system 700 of FIG. 10A. However, the advantage of the system 700 is that it can operate not only in the transmissive illumination mode but also in the reflective mode, and any geometric arrangement, such as, for example, a cylindrical geometry of free space (i.e., non-contact and no matching fluid). Can be satisfied.

Referring to FIG. 10B, the same reference numerals are used for the same members as those shown in FIG. 10A, and a third light source 760 is provided to make the light source much clearer. In some embodiments of the invention, the third light source 760 is at least two axes 762, 764, for example, if both side lighting with the second light source 726 and front illumination with the third light source 760 are provided. Can optionally move along

Side lighting and front lighting can be used to improve the concentration efficiency of low-level light signals, especially in perspective lighting modes using the first light source 719. Use for signals that are hidden by Measurements from the first, second and third light sources 719, 726, and 760 can be combined with the tomography process individually and used to solve transmission problems similar to those described above in connection with FIG. .

Referring to FIG. 11A, another imaging system 800 includes a laser source 802 and a single optical fiber 804 for generating excitation light. Optical fiber 804 is coupled with optical splitter 806, which emits excitation light into a plurality of optical fibers 808a through 808N and one optical fiber 810, each carrying excitation light. Optical fibers 808a-808N are connected with the scanning head 812, which can move in parallel along at least two axes indicated by arrows 814, 816. In a particular embodiment of the invention, the optical fibers 808a-808N provide a clear light source, which simultaneously projects excitation light towards the specimen 822 located within the imaging chamber 820 (or imaging chamber).

As described above, the fluorescence emitted by the internal light present in the sample 822 and the fluorescent protein in the sample 822 passes through the selection filter 828 and the image intensifier 830 to the CCD camera 832. do. The light is converted into digital data 832a by the CCD camera 832, and the digital data is received by the computer 834. Computer 834 processes the digital data and provides a graphical representation of the tomography image on graphical display 836, as described, for example, in process 200 of FIG.

The white light source 826 generates white light, which is reflected from the sample 822 and passes through the selection filter 828 and the image enhancer 830 to provide white light through the CCD camera. As described in connection with FIG. 4, the white light image of the sample 822 overlaps the map of the fluorescent protein in the sample 822 and results in a more understandable tomography image.

The computer 834 controls the position of the scanning head 812 through the XY-stage controller 838 and moves the scanning head about the axis indicated by arrows 814 and 816 to provide a clear light source for better monolayers of fluorescence. Create a shooting map.

In a particular embodiment of the invention, the scanning head 812 may be scanned along at least two axes indicated by arrows 814 and 816 and the optical fibers 808a to 808N connected with the scanning head 812 are all simultaneously. Can be illuminated. Advantages of such an embodiment include, but are not limited to, faster tomography imaging, especially when low amplitude optical signals result in long exposure times.

The crosstalk between the fibers 808a through 808N can be minimized by appropriately selecting the distance between the respective fibers 808a through 808N so that the paths of propagating photons do not overlap. In another embodiment of the invention, especially when the exposure time is short, the fibers 808a to 808N can be illuminated one at a time to remove noise due to crosstalk between the fibers 808a to 808N. In one embodiment of the present invention, optical splitter 806 may be replaced with an optical switch (eg, reference numeral 106 of FIG. 2). In another embodiment of the present invention, a single fiber may be scanned along the axis indicated by arrows 814 and 816.

Referring to FIG. 11B, using the same reference numerals for the same members as those shown in FIG. 11A, the scanning head 812 of FIG. 11B has a plurality of gaps 850a to 850N, each with a separate light source. Corresponding to N clear light sources. When the scanning head 812 moves along the axis indicated by arrows 814 and 816, it forms another distinct light source, for example N clear light sources.

The methods and systems of the present invention can use any fluorescent protein, including but not limited to DsRed and HcRed fluorescent proteins. Such specific fluorescent proteins provide fluorescence in the red or near infrared region in the visible spectrum. Because the red region of visible light has a higher efficiency at deeper penetration depth in biological tissues compared to other wavelengths of visible light, it is possible to generate a map of high quality fluorescent protein, which is higher in longer wavelength near-infrared (NIR) systems. The specific protein can map a higher quality fluorescent protein because it can provide resolution.

The methods and systems according to the invention can be used with cancer cells expressing fluorescent proteins (such as GFP) to study cancer growth and cancer metastasis formation.

The methods and systems according to the invention can be used with GFP expressing cancer cells and YFP expressing viral cells for gene delivery studies and gene therapy for targeted treatment of particular patients.

The method and system according to the present invention employ an imaging modality that uses computation for imaging of any geometric arrangement without the need for a matching fluid. Operations for modeling light propagation and solving transmission problems can be applied to both embodiments of the system.

The method and system according to the present invention provide higher spatial resolution when using visible light than the approach of conventional tomography using near infrared.

The resulting radiant fluorescence provided by the excitation light and the above described embodiments of the invention may be continuous wavelength (CW) light, intensity modulated (IM) or time resolved (TR) light, or a combination of both. The method and system of the present invention give information about the dynamics of the system as a function of time and the resulting image is obtained by other imaging methods such as magnetic resonance imaging (MRI), computed tomography (CT), ultrasound or bioluminescence imaging. It can be recorded with the obtained image.

The systems and methods described above use a modified diffusion approximation in combination with appropriate normalization, which is at least 400 nm in a medium having a relatively high absorption coefficient (eg> 0.3 cm ⁻¹ ), ie in a diffused medium. It enables three-dimensional tomography imaging of fluorescent proteins in vivo in the visible wavelength range of about 700 nm. The modified diffusion approximation does not need to use more complex transfer equations. Therefore, the modified solution is computationally efficient.

In one embodiment of the invention described above, non-contact tissue illumination and / or non-contact photoreception are used, and the apparent light source and / or light detector described above is placed away from the sample to be scanned. In another embodiment of the present invention, the apparent light source and / or light detector is placed in substantial contact with the sample.

The methods and systems described above can be applied to a variety of biological and molecular processing processes by using a variety of different fluorescent proteins. For example, in various embodiments, fluorescent proteins can be used to monitor cancer growth, metastasis formation, gene expression and therapeutic effects. In addition, the methods and systems according to the invention can be used to provide a non-invasive molecular image of the entire body that can non-invasively produce information related to subcellular levels of activity.

The methods and systems according to the present invention provide insight into specific molecular biological abnormalities that form the basis of many diseases such as cancer, tumor growth and metastasis formation. The methods and systems according to the present invention can also be used to image angiogenesis because the high uptake of hemoglobin contrasts the blood vessels against the fluorescent background of cancer cells. The methods and systems can also be used to obtain the efficacy of inherent targeted therapies at the molecular level. This, in turn, can influence drug development, drug testing and the selection of appropriate treatments for patients and treatment changes. The methods and systems also allow for the study of the origin of disease in the intact microenvironment of living systems. The methods and systems are also useful for testing unique gene delivery strategies. The imaging methods and systems allow for much faster three-dimensional acquisition of information than was possible with recent scientific techniques of time consuming and much effort.

The methods and systems according to the invention can be applied to a variety of biological, immunological and genetic therapies designed to promote the inhibition and eradication of various types of diseases including cancer, neurological diseases, inflammatory and infectious diseases. In addition, the methods and systems according to the present invention can be applied to a variety of applications for woundless disease detection and treatment in a combined set.

Although described with reference to the preferred embodiments of the present invention, those skilled in the art that various modifications and changes to the present invention without departing from the spirit and scope of the invention described in the claims below I can understand that you can.

Claims (43)

Includes a clear light source modified to project excitation light towards a sample having a fluorescent protein therein, And wherein the excitation light enters the sample and becomes internal light within the sample, the internal light is modified to excite fluorescence from the fluorescent protein, and the internal light and the fluorescence are diffused. The optical tomography system of claim 1, wherein at least one of the excitation light and the fluorescence has a wavelength in a visible light region. The optical tomography system of claim 1, wherein the fluorescence has a wavelength in the visible light region. The optical tomography system of claim 1, wherein the fluorescence has a wavelength in the red portion of the visible region. The optical tomography system of claim 1, wherein the fluorescence has a wavelength in the near infrared region. The method of claim 1, Modified to receive the internal light exiting the sample, modified to receive the fluorescence exiting the sample, further modified to convert the received internal light into first image information and converting the received fluorescence into second image information A photo detector further modified to convert And A light propagation model coupled with the light detector, the model is modified to predict light propagation in a diffusion medium, and further modified to combine the first image information, the second image information and the light propagation model, And an image processing apparatus further modified to provide an image of the fluorescent protein. 7. The optical tomography system of claim 6, wherein said image processing apparatus comprises a diffusion equation processing apparatus using a diffusion equation having a modified diffusion coefficient associated with at least one of said internal light and said fluorescence. 7. The optical tomography system of claim 6, wherein the light detector is selectively movable to receive the internal light and the fluorescence on multiple light paths for the sample. 7. The optical tomography system of claim 6, further comprising an optical scanner that provides the internal light and the fluorescence to the photo detector on a plurality of optical paths to the sample. The optical tomography system of claim 1, wherein the apparent light source further comprises a light indicating device capable of selectively moving the apparent light source directed toward the excitation light onto a plurality of optical paths directed to the specimen. . 12. The optical tomography system of claim 10, wherein the light directing device further comprises an optical switch capable of selectively moving the apparent light source providing a plurality of light paths toward the specimen. 12. The optical tomography system of claim 10, wherein the light indicating device comprises a mirror capable of selectively moving the apparent light source providing a plurality of light paths toward the specimen. 12. The optical tomography system of claim 10, wherein the light indicating device is modified to selectively translate the apparent light source along at least one of the apparent light source translational axes. The optical tomography system of claim 1, wherein the specimen is selectively moved to provide the excitation light over a plurality of optical paths to the specimen. 15. The optical tomography system of claim 14, wherein the sample is capable of selectively rotating about a sample axis of rotation. 15. The optical tomography system of claim 14, wherein the specimen is capable of selectively parallel translation along at least one of the specimen translation axis. The optical tomography system of claim 14, wherein the sample is capable of selectively rotating about a sample axis of rotation, and the sample is capable of selectively paralleling along at least one of the sample translation axis. 10. The apparatus of claim 1, wherein the apparent light source comprises a light indicating device capable of selectively moving the apparent light source directing the excitation light onto a plurality of optical paths directed to the sample, wherein the sample selectively moves. Thereby providing the excitation light on a plurality of optical paths to the specimen. The optical tomography system of claim 1, wherein the internal light passes through the sample with transmitted illumination. The optical tomography system of claim 1, wherein the internal light is reflected from the sample with reflected light. Generating an excitation light from an apparent light source to project light toward a sample having a fluorescent protein therein, Wherein the excitation light enters the sample and becomes internal light within the sample, the internal light is modified to excite fluorescence from a fluorescent protein, and the internal light and the fluorescence are diffused. 22. The optical tomography method of claim 21, wherein at least one of the excitation light and the fluorescence has a wavelength in the visible light region. 22. The method of claim 21, wherein the fluorescence has a wavelength in the visible light region. 22. The method of claim 21, wherein the fluorescence has a wavelength in the red portion of the visible light region. 22. The optical tomography method of claim 21, wherein the fluorescence has a wavelength in the near infrared region. The method of claim 21, Receiving the internal light exiting the sample; Receiving the fluorescence exiting the sample; Converting the received internal light into first image information; Converting the received fluorescence into second image information; Generating a model modified to anticipate light propagation in the diffusion medium; And And combining the first image information, the second image information, and the model to provide an image of the fluorescent protein. 27. The method of claim 26, wherein receiving the interior light and receiving the fluorescence comprise the interior with a selectively moveable photo detector modified to receive the interior light and the fluorescence on multiple light paths to the sample. And receiving light and said fluorescence. 27. The method of claim 26, wherein the model is generated according to a solution to a diffusion equation having a modified diffusion coefficient related to at least one of the internal light and the fluorescence. 22. The method of claim 21, further comprising selectively moving the apparent light source that directs the excitation light onto a plurality of optical paths toward the specimen. 30. The method of claim 29, wherein the apparent light source comprises an optical switch capable of selectively moving the apparent light source. 30. The method of claim 29, wherein the apparent light source comprises a mirror capable of selectively moving the apparent light source. 30. The method of claim 29, wherein selectively moving the apparent light source comprises selectively moving the apparent light source along at least one apparent light source translation axis. 22. The method of claim 21, further comprising selectively moving the sample to provide the excitation light over a plurality of optical paths to the sample. 34. The method of claim 33, wherein selectively moving the sample comprises selectively rotating the sample about a sample axis of rotation. 34. The method of claim 33, wherein selectively moving the sample comprises selectively moving the sample along at least one sample translation axis. The method of claim 33, wherein selectively moving the sample, Optionally rotating the specimen about a specimen axis of rotation; And And selectively moving the sample along at least one sample translation axis. The method of claim 21, Selectively moving the apparent light source that directs the excitation light onto a plurality of optical paths toward the specimen; And And selectively moving the sample to provide the excitation light over a plurality of different optical paths to the sample. 22. The method of claim 21, wherein the internal light passes through the sample with transmitted illumination. 22. The optical tomography method of claim 21, wherein the internal light is reflected from the sample with reflected light. And at least one selectively movable member for selectively moving an apparent light source directed toward the plurality of light paths toward the specimen. 41. The optical tomography system of claim 40, wherein the selectively movable member comprises at least one selectively movable mirror. 41. The optical tomography system of claim 40, wherein the selectively movable member comprises a selectively movable structure. 43. The optical tomography system of claim 42, further comprising optical fibers coupled to the selectively movable structure.

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